A pyridoxal phosphate–dependent enzyme that oxidizes an unactivated carbon-carbon bond

Abstract

Pyridoxal 5'-phosphate (PLP)-dependent enzymes have wide catalytic versatility but are rarely known for their ability to react with oxygen to catalyze challenging reactions. Here, using in vitro reconstitution and kinetic analysis, we report that the indolmycin biosynthetic enzyme Ind4, from Streptomyces griseus ATCC 12648, is an unprecedented O2- and PLP-dependent enzyme that carries out a four-electron oxidation of L-arginine, including oxidation of an unactivated carbon-carbon (C-C) bond. We show that the conjugated product of this reaction, which is susceptible to nonenzymatic deamination, is efficiently intercepted and stereospecifically reduced by the partner enzyme Ind5 to give D-4,5-dehydroarginine. Thus, Ind4 couples the redox potential of O2 with the ability of PLP to stabilize anions to efficiently oxidize an unactivated C-C bond, with the subsequent stereochemical inversion by Ind5 preventing off-pathway reactions. Altogether, these results expand our knowledge of the catalytic versatility of PLP-dependent enzymes and enrich the toolbox for oxidative biocatalysis.

Emergence of oxygen‐ and pyridoxal phosphate‐dependent reactions

“…The amino acid l ‐arginine has emerged as a common substrate for sequence‐related oxygen‐dependent PLP enzymes from Streptomyces bacteria. These enzymes include Ind4 from the indolmycin biosynthetic pathway [71,72], MppP from enduracididine biosynthesis [73,74], and RohP from the azomycin biosynthetic pathway [75,76]. Indolmycin, enduracididine, and azomycin each contain arginine‐derived heterocycles, for which Ind4, MppP, and RohP are essential upstream enzymes toward the biosynthesis of each heterocycle.…”

“…Following ring formation, the exocyclic amine is methylated. The production of d ‐4,5‐dehydroarginine is dependent on Ind4, an oxygen‐ and PLP‐dependent oxidase, and Ind5, a stereospecific NAD(P)H‐dependent dehydrogenase, respectively [72,79]. The double bond formed adjacent to the guanidine group is essential to the formation of the oxazolinone ring, as arginine cannot be used in this transformation.…”

In vitro Reconstitution of the Biosynthetic Pathway to the Nitroimidazole Antibiotic Azomycin

“…While PLP-dependent enzymes are extremely diverse,t heir reactions with oxygen are generally limited to so-called paracatalytic reactions that produce unintended side products in low amounts. [15] Our interest in O 2 -, PLP-dependent enzymes recently led us to identify at hird O 2 -, PLP-dependent arginine oxidase RohP from Streptomyces cattleya DSMZ 46 488. These include decarboxylases,such as plant phenylacetaldehyde synthase, [12] as well as the l-arginine oxidases MppP from l-enduracididine biosynthesis [13,14] and Ind4 from indolmycin biosynthesis (Supporting Information, Figure S2).…”

Structural Basis for Phospholyase Activity of a Class III Transaminase Homologue

“…4.2.3.2) activity towards O-phosphoethanolamine (PEtN), an activity described previously for vertebrate enzymes such as human AGXT2L1, enzymes for which no structure has yet been reported. In order to shed light on the distinctive features of PLP-dependent phospholyases, structures of A1RDF1 in complex with PLP (internal aldimine) and PLP·PEtN (external aldimine) were determined, revealing the basis of substrate binding and the structural factors that distinguish the enzyme from class III homologues that display TA activity.Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a wide range of chemical reactions, including the racemisation and decarboxylation of amino acids and transamination between amino acid donors and keto acid acceptors, [1,2] and new reactions, including oxidations, [3] continue to be discovered. Some of these enzymes, notably those of the "PLP fold type I" and belonging to the class III transaminase (TA) subgroup, have become extremely useful in biotechnology, because some members possess the ability to form chiral amines from ketone precursors, [4] whereas others catalyse the useful racemisation of amino acid amides.…”

“…Pyridoxal-phosphate (PLP)-dependent enzymes catalyse a wide range of chemical reactions, including the racemisation and decarboxylation of amino acids and transamination between amino acid donors and keto acid acceptors, [1,2] and new reactions, including oxidations, [3] continue to be discovered. Some of these enzymes, notably those of the "PLP fold type I" and belonging to the class III transaminase (TA) subgroup, have become extremely useful in biotechnology, because some members possess the ability to form chiral amines from ketone precursors, [4] whereas others catalyse the useful racemisation of amino acid amides.…”